Selection And Analysis Of Different Reference Genes In Tumor Gene Expression

Quantitative real-time polymerase chain reactions (qRT-PCR) has become a rapid, sensitive and quantitative method to measure RNA transcript abundance. Useful data from this method depend on fitting data to theoretical curves that allow computation of mRNA levels. Calculating accurate mRNA levels requires important parameters such as reaction efficiency and the fractional cycle number at threshold (CT) to be used; however, the amplification efficiency, changing during PCR, may be overestimated by the traditional standard curve method. No single gene meets the criteria of an ideal endogenous reference. Therefore, it is necessary to select suitable reference genes for specific requirements.The present study evaluated5potential reference genes in the human Lung cancer(42cases), Gastric cancer(35cases), Breast cancer(29cases), Colon cancer(42cases) and Rectal cancer(44cases) using three different statistical algorithms, geNorm, NormFinder, and BestKeeper. On qRT-PCR data analyses using a completely objective and noise-resistant algorithm,5reference genes met standard efficiency criteria. Validation of their stability and suitability as reference genes using geNorm suggested RPLPO as the most stable one in lung cancer, GAPDH, ACTB and RPLPO as the most stable ones in Rectal cancer,ACTB, RPLPO and TFRC as the most stable ones in Gastric cancer, ACTB as the most stable one in Breast cancer and ACTB as the most stable one in Colon cancer; NormFinder indicated that RPLPO in lung cancer, GAPDH in Rectal cancer, ACTB in Gastric cancer, ACTB in Breast cancer, ACTB in Colon cancer had the highest expression stabilities, and BestKeeper highlighted RPLPO in lung cancer, GAPDH in Rectal cancer, GAPDH in Gastric cancer, ACTB in Breast cancer, GAPDH in Colon cancer as reference genes. Combining these three algorithms suggested the genes RPLPO in lung cancer, GAPDH, ACTB and RPLPO in Rectal cancer, ACTB, RPLPO and TFRC in Gastric cancer, ACTB in Breast cancer, ACTB in Colon cancer as stable endogenous references in qRT-PCR analysis of samples, with TFRC in lung cancer, Rectal cancer, Breast cancer and Colon cancer being the least stable one and GUSB being the least stable one in Gastric cancer.After the use of the geometric mean algorithm to obtain the mean Ct value of multiple reference genes, the interest gene is normalized. With the application of relative expression values, we analyze the differences and correlation between multi-genes in different tumors. Most genes show a significant difference in mRNA expression level among various tumors. The correlation analysis show that gene expression levels of STMN1, TYMS in the breast cancer and STMN1, TOP2A in gastric cancer are highly correlated, the correlation coefficients were:r=0.891, P <0.0001and r=0.824, P0.0001respectively. In addition, we charted the related genes according to the moderate correlation coefficient value that is greater than0.5. The reference gene selected in this project is accurate and appropriate after comparison analysis.